Model for mutually exclusive domain folding molecular switch

ABSTRACT

The present invention is a model for a mutually exclusive domain folding molecular switch comprising a two-domain, bifunctional fusion protein wherein the free energy released by the folding of a first domain of the fusion protein drives an unfolding of a second domain of the fusion protein, and vice versa. The molecular structure of the fusion protein is engineered so that, at any time, the folding of the first domain necessarily unfolds the other domain, and vice versa, thereby making the folded and unfolded states of the first and second domains mutually exclusive. This is accomplished by the insertion of an exemplary insert protein into a surface loop of an exemplary target protein subject to a novel structural design criterion wherein the N-C terminal length of the exemplary insert protein is at least two-times greater than the Cα-Cα length of the surface loop of the exemplary target protein.

BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The invention relates generally to a model for a molecular switchto modulate the bioactivity of proteins

[0003] 2. Related Art

[0004] Ribonucleases are hydrolase enzymes that break linkages betweennucleotides in ribonucleic acid. They are accordingly highly cytotoxic.A major problem with their use as therapeutic agents, for example, aspharmacologic agents in the treatment of cancer, is that theircytotoxicity is indiscriminate. Currently available ribonucleasepharmacologic agents kill normal as well as neoplastic cells, and theside effects of their use can be severe. Additionally, currentlyavailable ribonuclease agents demonstrate poor bioavailability owing totheir rapid degradation by the liver and their difficulty in passingthrough both normal and neoplastic cell membranes.

SUMMARY OF THE INVENTION

[0005] The present invention comprises a model for a mutually exclusivefolding domain molecular switch (and a method for the creation thereof),which model includes a fusion protein comprising at least one insertprotein having an insert domain lying between an amino terminal and acarboxyl terminal of the at least one insert protein, the insert domainbeing associated with a first quantity of free energy; and, a targetprotein having at least one surface loop that begins at an alpha carbonof a first surface loop amino acid and terminates at an alpha carbon ofa second surface loop amino acid, the at least one surface loopcomprising a target domain of the target protein, the target domainbeing associated with a second quantity of free energy, wherein, the atleast one insert protein is operatively inserted within the at least onesurface loop between the alpha carbon of first surface loop amino acidand the alpha carbon of second surface loop amino acid such that anamino-carboxyl length extending between an alpha carbon of the aminoterminal of the at least one insert protein and an alpha carbon of thecarboxyl terminal of the at least one insert protein is at leasttwo-times greater than an alpha-carbon-alpha-carbon length extendingbetween the alpha carbon of the first surface loop amino acid and thealpha carbon of the second surface loop amino acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1A is a schematic illustration of a domain of an exemplaryinsert protein in an unfolded conformation.

[0007]FIG. 1B is a schematic illustration of a domain of an exemplaryinsert protein in an folded conformation.

[0008]FIG. 1C is a schematic illustration of a conformation of anexemplary target protein having a folded domain in the shape of a wedgeand having a surface loop.

[0009]FIG. 1D is a schematic illustration of a conformation of anexemplary target protein having an unfolded domain in the shape of astraight line and having a surface loop.

[0010]FIG. 1E is a schematic illustration of a exemplary fusion proteincapable of existing in two mutually exclusive conformations.

[0011]FIG. 1F is a schematic illustration of a exemplary fusion proteincapable of existing in two mutually exclusive conformations, in which anequilibrium state has been influenced by the binding of a ligand.

[0012]FIG. 2A is a schematic illustration of a human ubiquitin molecule.

[0013]FIG. 2B is a schematic illustration a barnase molecule.

[0014]FIG. 3 is a graph of circular dichroism spectra of aubiquitin-barnase fusion protein as a function of temperature, showingtemperature-induced conformational change in the structure ofubiquitin-barnase fusion protein.

[0015]FIG. 4 is a graph of the conversion an ubiquitin-barnase fusionprotein from the barnase conformation to the ubiquitin conformation,monitored by ellipticity at 230 nm (open triangles, solid line) andbarnase enzymatic activity (closed triangles, dashed line). Lines arefor illustrative purpose only.

[0016]FIG. 5 is a graph showing the denaturation of the barnase domain(10° C., circles) and the ubiquitin domain (40° C., squares) induced byurea and guanidine hydrochloride (“GdnHCl”), respectively.

[0017]FIG. 6 is a graph of a circular dichroism spectral recording at 40degrees C., showing barstar-induced folding of the barnase domain andunfolding of the ubiquitin domain.

[0018]FIG. 7 is a graph of a circular dichroism spectral recording at 15degrees C., showing barstar-induced folding of the barnase domain andunfolding of the ubiquitin domain.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The model for a mutually exclusive domain folding molecularswitch is a novel and powerful approach to understanding the fundamentalmechanisms of allosteric switching in molecular biology and for thedeveloping diagnostic and therapeutic proteins with novel capabilities,possessing the following advantages:

[0020] [i] mechanism of the molecular switch it is inherentlycooperative; and,

[0021] [ii] the all-or-nothing action of the mechanism of the molecularswitch assures that it behaves in a binary fashion; and,

[0022] [iii] the switching mechanism is reversible; and,

[0023] [iv] the position of a reciprocally folding and unfoldingconformational equilibrium, to be described hereinafter, can be readilyadjusted by external factors.

[0024] In their simplest form, proteins are polypeptides, i.e., linearpolymers of amino acid monomers. However, the polymerization reactionwhich produces a polypeptide results in the loss of one molecule ofwater from each amino acid. Consequently, a polypeptide is morerigorously defined as a polymer of amino acid residues. Natural proteinmolecules may contain as many as 20 different types of amino acidresidues, each of which contains a distinctive side chain.

[0025] An amino acid is an organic molecule containing an amino group(“—NH₂”) and a carboxylic acid group (“—COOH”). While there are manyforms of amino acids, all of the important amino acids found in livingorganisms are alpha-amino acids. Alpha amino acids have their both their—COOH and —NH₂ groups attached to the same carbon atom, which is calledthe alpha carbon atom.

[0026] Thus, all of the important amino acids found in living organismsconsist of an alpha carbon atom to which there is attached:

[0027] [i] A hydrogen atom

[0028] [ii] An amino group (—NH₂)

[0029] [iii] A carboxyl group (—COOH).

[0030] [iv] One of 20 different “R” groups.

[0031] It is the structure of the R group that distinguishes each aminoacid structurally and determines its biochemical properties. Moreover,the structure and biochemical properties of a protein is dictated by theprecise sequence of the amino acids in the polypeptide chains of whichit is comprised

[0032] The particular linear sequence of amino acid residues in thepolypeptide chain comprising a protein defines the primary structure ofthat protein. However individual polypeptides and groups of polypeptidesundergo spontaneous structural alteration and association into a numberof recurring intermediate patterns such as, for example, helices,including alpha helices, and sheets, including beta sheets. Theserecurring intermediate polypeptide patterns are referred to as aprotein's secondary structure. The spontaneous structural alteration andassociation of polypeptide chains into a secondary structure isdetermined by the sequence of amino acids in the polypeptide chains andby the ambient biochemical environment.

[0033] The helices, sheets, and other patterns of a protein's secondarystructure additionally undergo a process of thermodynamically-preferredcompound folding to produce a three-dimensional or tertiary structure ofthe protein. The fully folded conformation of the protein is maintainedby relatively weak interatomic forces such as, for example, hydrogenbonding, hydrophobic interactions and charge-charge interactions.Covalent bonds between sulphur atoms may also participate in proteinfolding into a tertiary conformation by forming intra-moleculardisulfide bridges in a single polypeptide chain, as well as by formingintermolecular disulfide bridges between separate polypeptide chains ofa protein. This ability of polypeptide chains to fold into a greatvariety of structures, combined with the large number of amino acidsequences of a polypeptide chain that can be derived from the 20 commonamino acids in proteins, confers on protein molecules their great rangeof biological activity.

[0034] The tertiary structure of a protein may contain a surface loop. Asurface loop is a continuous length of polypeptide chain whoseconstituent amino acids are in neither an alpha helical conformation norin a beta sheet conformation, and can contact at least five watermolecules, as determined by the DSSP computer program of Wolfgang Kabschand Chris Sander. The DSSP, a program which is well known in the art,defines secondary structure, geometrical features and solvent exposureof proteins, given atomic coordinates in Protein Data Bank format, whichis also well known in the art. (W. Kabsch & C. Sander, “Dictionary ofprotein secondary structure: pattern recognition of hydrogen-bonded andgeometrical figures”, Biopolymers 22, 2577-2637. (1983); See also,Centre for Molecular and Biomolecular Informatics, University ofNijmegen, Toernooiveld 1, P.O. Box 9010, 6500 GL Nijmegen, +31(0)24-3653391, postmaster@cmbi.kun.nl; http://www/cmbi.kun.nl.gv/dssp/)

[0035] Protein folding occurs on a global level that endows the entireprotein molecule with a three dimensional structure and surfacetopology. Protein folding also occurs at a local level at multiple sitesupon and within a protein. Locally, folding may involve one or morepolypeptide subunits of the protein to endow different regions of theprotein with different specific biological activities, or differentspecific molecular architectures, such as, for example, fashioning alocation in a protein molecule into a receptor site for anothermolecule.

[0036] As used herein, the term domain means the molecular structure ofan entire protein molecule or the molecular structure of a part,portion, or region of the molecular structure of a protein molecule,including a part, portion, or region of the protein molecule's surfaceor the protein molecule's interior. A domain may refer only to adistinction in a protein molecule's structure, such as for example, analpha helix or a beta sheet. A domain may or may not have an associatedbiological function, such as a regulatory, receptor, signaling, active,catalytic, or other biological function. A domain may further beassociated with a free energy, i.e., a thermodynamic state function thatindicates the amount of energy that stabilizes the domain when theprotein, or part thereof, with which the domain is associated is in afolded configuration. All of part of the free energy may be availablefor the domain to do biochemical work.

[0037] Because the folding of a protein molecule is both a global andlocal process, it can endow a protein molecule with both global andlocal structural and biological properties, such as, for example, anenzymatic activity, or a capacity and specificity for binding otherproteins, such as antigens. Consequently, the biological functions of aprotein depend on both its global folded tertiary structure, which isalso called its native or folded conformation, as well as the foldedstructure of regions of the protein. Conversely, a global or localunfolding of a protein deactivates its global or local biologicalactivity. An unfolded, biologically inactive protein is said to be in adenatured or unfolded conformation.

[0038] Many proteins are comprised of domains that communicate with eachother by means of conformational changes in the structure of the proteinof which they are a part, in order to activate or deactivate abiological function. For example, in the case of a protein that is anenzyme, ligand binding or phosphorylation can serve as a switchingmechanism to induce structural changes within the enzyme's regulatorydomain, which then triggers activity in the enzyme's catalytic domain.

[0039] Another type of switching mechanism is illustrated in vivo byproteins that are unfolded in physiological conditions but fold uponbinding to a cellular target. In this molecular switching mechanism, thefolding and unfolding of a regulatory domain of a protein modulates thefunction of the protein via propagation of structural changes to itsactive domain.

[0040] In the case of the protein serving as an exemplary embodiment ofthe model for a mutually exclusive domain folding (hereinafter, “MEDF”)switch, the protein is a exemplary fusion protein synthesized from:

[0041] [i] an exemplary insert protein having an insert domain lyingbetween an amino terminal and a carboxyl terminal, which insert domainis associated with a first quantity of free energy; and,

[0042] [ii] an exemplary target protein having at least one surface loopthat begins at an alpha carbon of a first surface loop amino acid andterminates at an alpha carbon of a second surface loop amino acid, whichsurface loop comprises a target domain associated with a second quantityof free energy.

[0043] The amino terminal of the exemplary insert protein is spatiallyseparated from the carboxyl terminal of the exemplary insert protein bya linear distance known as the amino-carboxyl length (hereinafter, the“N-C terminal length”) of the exemplary insert protein, that is measuredwhen the exemplary insert protein is in its folded conformation. Thefirst surface loop amino acid of the exemplary target protein isspatially separated from the second surface loop amino acid of theexemplary target protein by a linear distance known as thealpha-carbon-alpha-carbon length of the surface loop of the exemplarytarget protein (hereinafter, the “Cα-Cα length”), that is also measuredwhen the exemplary target protein is in its folded conformation.

[0044] The molecular structure of the exemplary fusion protein isengineered so that, at any time, the folding of the insert domainnecessarily unfolds the target domain, and vice versa, thereby makingthe folded and unfolded states of the insert and target domains mutuallyexclusive. This mutual exclusion of concurrently folded or concurrentlyunfolded states is accomplished by the insertion of the exemplary insertprotein into the surface loop of the exemplary target protein subject toa novel structural design criterion wherein the N-C terminal length ofthe exemplary insert protein is at least two-times greater than theCα-Cα length of the surface loop of the exemplary target protein.

[0045] The present invention is an exemplary embodiment of a model for amutually exclusive domain folding (“MEDF”) molecular switch comprisingan exemplary two-domain, bifunctional fusion protein, wherein the freeenergy released by the folding of a first domain of the exemplary fusionprotein drives unfolding of a second domain of the exemplary fusionprotein, and vice versa.

[0046] Subject to this novel structural design criterion, a dynamicstate of thermodynamic and structural equilibrium is established in theexemplary fusion protein that disenables the insert domain of theexemplary insert protein and the target domain of the exemplary targetprotein from simultaneously co-existing in their native folded states.

[0047] Accordingly, any excess free energy present in one of the twodomains that is not necessary to stabilize its folded configuration isspontaneously transferred, through the structure of the exemplary fusionprotein, to the other of the two domains to unfold it from its foldedconfiguration, and vice versa. In effect, the excess free energy storedin the folded conformation of one domain is used to drive the unfoldingof the other domain; and, the molecular structure of the exemplaryfusion protein is engineered to create a dynamic state of thermodynamicand correlative structural equilibrium that is determined by therelative thermodynamic and structural stabilities of the two domains.

[0048] Viewed another way, the molecular structure of the exemplaryfusion protein is engineered to create a MEDF molecular switch bycreating cooperatively folding-unfolding subunits comprising two proteindomains, which two domains cannot simultaneously exist in their foldedstates. This scheme is depicted in FIGS. 1A-F

[0049] In FIG. 1A, exemplary insert protein 51, having an amino terminal21 and a carboxyl terminal 22, exists in an unfolded conformation 20,thereby forming an exemplary unfolded insert domain, schematicallyillustrated as a hatched ribbon that is coincident with the extent ofexemplary insert protein 51. In FIG. 1B, exemplary insert protein 51,having an amino terminal 21 and a carboxyl terminal 22, exists in afolded conformation 23, thereby forming an exemplary folded insertdomain, schematically illustrated as a hatched double-crossed ribbonthat is coincident with the extent of exemplary insert protein 51, andfolds to form indentation 24.

[0050] In FIG. 1C, there is shown schematically a folded conformation 26of an exemplary target protein 41 having an exemplary folded targetdomain in the shape of a wedge 46. Exemplary target protein 41 also hasa surface loop 27, schematically shown as a nearly full circle, arisingfrom a first alpha carbon forming a first terminus 28 of a first arm 29of wedge 46, and ending at a second alpha carbon forming a secondterminus 30 of a second arm 31 of wedge 46.

[0051] Also shown schematically in FIG. 1C is line 45, representing theCα-Cα length of the surface loop 27.

[0052] In FIG. 1D, there is shown schematically an unfolded conformation32 of exemplary target protein 41 in which exemplary folded targetdomain 46 has unfolded into the shape of straight line 56. Unfoldedconformation 32 of exemplary target protein 41 also has surface loop 27,now shown as a half-circle arising from first alpha carbon forming afirst terminus 28 of the straight line 56 and ending at a second alphacarbon forming a second terminus 30 of the straight line 56.

[0053] In FIG. 1E, there is shown schematically an exemplary fusionprotein 35 comprised of exemplary insert protein 51 inserted intosurface loop 27 of exemplary target protein 41, which exemplary fusionprotein 35 is capable of existing in two mutually exclusiveconformations 35L and 35R, representing the mutually exclusive binarystates of the model for the MEDF molecular switch embodied in exemplaryfusion protein 35 .

[0054] The image to the left of the antiparallel arrows 36 of FIG. 1Eshows exclusive state 35L of exemplary fusion protein 35, whereinexemplary insert protein 51, with its exemplary insert domain inunfolded (hatched ribbon) conformation 20, (as shown in FIG. 1A), hasbeen inserted into surface loop 27 of exemplary target protein 41 withits exemplary target domain in its folded conformation 46, (as shown inFIG. 1C). The image to the right of the antiparallel arrows 36 of FIG.1E shows exclusive state 35R of exemplary fusion protein 35, whereinexemplary insert protein 51, with its exemplary insert domain in itsfolded (hatched double-crossed ribbon) conformation 23, (as shown inFIG. 1B), inserted into surface loop 27 of exemplary target protein 41with its exemplary target domain in its unfolded (straight line)conformation 56 (as shown in FIG. 1D).

[0055] In FIG. 1F, exemplary fusion protein 35 is again shownschematically existing in two mutually exclusive conformations 35L and35R, representing the mutually exclusive binary states of the model forthe MEDF molecular switch embodied in exemplary fusion protein 35.However the dynamic equilibrium existing between conformations 35L and35R has been shifted to the right by the binding of exemplary ligand 40to the indentation 24 of exemplary insert domain of protein 51 in foldedconformation 23.

[0056] If the exemplary insert domain of exemplary insert protein 51 infolded conformation 23 (FIG. B and FIG. 1E Right) is more stable thanexemplary target domain having wedge 46 of exemplary target protein 41in its folded conformation 26, (FIG. 1C and FIG. 1E Left), thenexemplary insert domain of exemplary insert protein 51 in foldedconformation 23 (FIG. 1B and FIG. 1E Right) will have an excess of freeenergy with which to forcibly stretch and unfold folded conformation 26of exemplary target domain, having wedge 46, of exemplary target protein41 (FIG. 1C and FIG. 1E Left), thereby unfolding wedge 46 into line 56,and yielding exemplary fusion protein 35 in state 35R.

[0057] If exemplary target domain, having wedge 46, of exemplary targetprotein 41 in folded conformation 26 (FIG. 1C and FIG. 1E Left) is morestable than exemplary insert domain of exemplary insert protein 51 infolded conformation 23 (FIG. 1B and FIG. 1E Right), then exemplarytarget domain, having wedge 46, of exemplary target protein 41 in itsfolded conformation 26 (FIG. 1C and FIG. 1E Left) will have an excess offree energy with which to forcibly stretch and unfold exemplary insertdomain of exemplary insert protein 51 in folded conformation 23 (FIG. 1Band FIG. 1E Right), thereby folding line 56 into wedge 46, and yieldingexemplary fusion protein 35 in state 35L.

[0058] In this manner, exemplary embodiment of the model for a MEDFswitch fully exploits the free energy stored in the folded conformationsof the aforementioned domains, as well as the inherent cooperativity ofreciprocal domain folding, to create a molecular switch of unprecedentedefficiency. Consequently, the model for a MEDF molecular switch is anovel and powerful approach to understanding the fundamental mechanismsof allosteric switching in molecular biology and for the developingdiagnostic and therapeutic proteins with novel capabilities, possessingthe following advantages:

[0059] [i] mechanism of the molecular switch it is inherentlycooperative; and,

[0060] [ii] the all-or-nothing action of the mechanism of the molecularswitch assures that it behaves in a binary fashion; and,

[0061] [iii] the switching mechanism is reversible; and,[iv] theposition of the folding/unfolding equilibrium can be readily adjusted byexternal factors.

[0062] For example, When present at 1 μM concentration, a ligand thatbinds to a protein with a dissociation constant of 1 nM will stabilizethe native conformation of the protein by as much as RT °1n(10³), or 4.2kcal mol¹ at 37 degrees C. This value is comparable to the total freeenergy change of folding for many proteins.

[0063] While the exemplary embodiment of the model for a MEDF switchentails the creation of a two-domain, bifunctional exemplary fusionprotein to be described more fully hereinafter, the exemplary embodimentof the model for a MEDF switch disclosed herein is not limited to theinsertion of an exemplary insert protein into a exemplary target proteinhaving only one domain or only one biological function. The exemplaryembodiment of the model for a MEDF switch disclosed herein comprisescases wherein one or more exemplary insert proteins is inserted into oneor more surface loops of exemplary target proteins having multipledomains and multiple biological functions, the effect of theseinsertions being to form a one or more cooperatively folding-unfoldingsubunits in the resultant exemplary fusion protein, each comprising twoprotein domains, which two domains cannot simultaneously exist in theirfolded states, thereby forming one or more cooperative, reversible, MEDFmolecular switches in the same exemplary fusion protein, each of whichis responsive to different controllable effector signals such as, forexample, ligand binding, pH, temperature, chemical denaturants, or thepresence of stabilizing or destabilizing mutations in either the barnaseor ubiquitin domains.

[0064] To create a specific nonlimiting exemplary embodiment of themodel for a MEDF switch, the inventors herein created a two-domainbifunctional fusion protein comprising the insertion of an exemplaryinsert protein, the human ubiquitin molecule having one regulatorydomain, into a selected surface loop of an exemplary target protein,barnase, which has one catalytic (or cytotoxic) domain.

[0065] The exemplary insert protein, human ubiquitin, is a proteinhaving one regulatory domain, and one biological function, that ofserving as a signaling marker or flag.

[0066] The exemplary target protein, barnase is a ribonuclease producedexclusively by the bacterium Bacillus amyloliquefaciens. Barnase has onecatalytic domain that is functionally cytotoxic to all mammalian celltypes.

[0067]FIG. 2A is a schematic illustration a molecule of human ubiquitin.The sheet-like arrows and ribbons in FIG. 2A represent beta strands andalpha helices, respectively. The noodle-like strands in the protein areloops and turns of the ubiqutin molecule. In FIG. 2A, the N-C terminallength from ubiquitin is about 38 Å in its folded conformation, asindicated by the double-headed arrow bearing the bearing the legend “38Å.

[0068]FIG. 2B is a schematic illustration a barnase molecule. Thesheet-like arrows and ribbons in FIG. 2A represent beta strands andalpha helices, respectively. The noodle-like strands in the protein areloops and turns of the barnase molecule. The barnase molecule has asurface loop in which the Cα-Cα length, measured from alpha-carbon ofthe loop amino acid proline in the number 64 position (“Pro64”) to thealpha-carbon of the loop amino acid threonine in the number 70 position(“Thr70”) is about 10.4 Å, with the barnase molecule in its foldedconformation, as indicated by the double-headed arrow bearing the legend“10.4 A.” The asterisk in FIG. 2B represents the point at which theubiquitin molecule, shown in FIG. 2A is inserted between amino acidresidue 66 and amino acid residue 67 of barnase.

[0069] The exemplary insert protein ubiquitin and the exemplary targetprotein barnase satisfy the novel structural design criterion that theN-C terminal length of the exemplary insert protein be at least twicethe Cα-Cα length of the exemplary target protein surface loop selectedfor insertion. In the exemplary fusion protein resulting from theinsertion of the exemplary insert protein ubiquitin molecule into thesurface loop of the exemplary target protein barnase, the foregoingstructural design criterion is satisfied.

[0070] Consequently, the regulatory domain of ubiquitin and thecatalytic domain of barnase cannot simultaneously co-exist in theirfolded states; and, the regulatory domain of ubiquitin, may be used toregulate the cytotoxic activity of barnase. Moreover, the ubiquitin andbarnase domains participate in a cooperative and reversibleconformational equilibrium, that may be influenced and controlled by avariety of controllable effector signals such as, for example, ligandbinding, pH, temperature, chemical denaturants, or the presence ofstabilizing or destabilizing mutations in either the barnase orubiquitin domains.

[0071] The ubiquitin and barnase genes are created by annealing andligating synthetic oligonucleotides (Integrated DNA Technologies)according to standard protocols.

[0072] The ubiquitin-barnase fusion gene is made by first adding anexemplary five amino acid linker (Gly-Thr-Gly-Gly-Ser) between the Lys66and Ser67 codons of the barnase gene. The inserted DNA containsexemplary KpnI and BamHI restriction sites that are used to introducethe ubiquitin gene.

[0073] The exemplary five amino acids of the exemplary linkerindividually serve as short, flexible linkers at the points ofattachment. The ubiquitin gene is inserted between the Thr and Glycodons of the linker.

[0074] All genes are fully sequenced to verify their integrity.

[0075] An interim ubiquitin-barnase fusion expression plasmid pETMT iscreated by using exemplary NdeI and XhoI enzymes to insert theubiquitin-barnase fusion gene into a plasmid, such as, for example, apET25b(+) plasmid (Novagen), or any other T7 promotor-containing plasmidthat also confers resistance to an antibiotic other than ampicillin.

[0076] In order to make the plasmid stable in E. coli, the gene forbarstar, the intracellular inhibitor of barnase that is coexpressed withbarnase by Bacillus amyloliquefaciens (together with its naturalpromoter from Bacillus amyloliquefaciens), is cleaved out of anexemplary pMT1002 plasmid (gift of Dr. Y. Bai, National Institutes ofHealth), or any other T7 promotor-containing plasmid that also confersresistance to an antibiotic other than ampicillin, with ClaI and PstIenzymes. The barstar gene is then placed between Clal and PstIrestriction sites on the pETMT plasmid (prior to this step, these sitesare introduced using the QuikChange mutagenesis kit (Strategene)).

[0077] In order to obtain milligram quantities of the ubiquitin-barnasefusion protein, it is necessary to increase cellular levels of barstarand purify the inactive ubiquitin-barnase fusion-barstar complex.Accordingly, the barstar gene is cloned into an exemplary pET41 plasmid(Novagen), thereby placing it under control of a T7 promoter andconferring upon the transformed cells resistance to kanamycin or anyother antibiotic other than ampicillin.

[0078]E. coli BL21 (DE3) cells are transformed with both plasmids, grownin a temperature range between about 20 degrees C. and 37 degrees C. inexemplary Luria-Bertani medium containing ampicillin and kanamycin toOD₆₀₀=1.0, and induced with 100 mg/L IPTG. Bacteria are harvested about2 to 12 hours later by centrifugation.

[0079] Cells are lysed in about 10 mM sodium phosphate (pH 7.5) byrepeated freeze-thaw cycles in the presence of a small amount oflysozyme at a concentration of about lysozyme is 10 mg/liter. ExemplaryDNase I (Sigma) at a concentration of about 10 mg/liter is then added toreduce viscosity, and the solution is centrifuged to remove insolubles.8 M urea is added to the supernatant to dissociate bound barstar, whichis subsequently removed by passing the solution through DE52 resin(Whatman) or a substantially equivalent anion exchange chromatographyresin. The solution is then loaded onto a HiTrap heparin column(Amersham-Pharmacia) or substantially equivalent cation exchange column,washed with 10 mM sodium phosphate (pH 7.5) and 6 M urea, and elutedwith a 0-0.2 M NaCl gradient.

[0080] Western blot analysis using anti-ubiquitin antibodies is used toshow that the major impurities are truncated ubiquitin-barnase fusionprotein products in which the ubiquitin domain, which is unfolded in theubiquitin-barnase fusion protein-barstar complex, is partially digested.These proteins, however, elute significantly later than the intactubiquitin-barnase fusion protein in the NaCl gradient. The urea isremoved by dialysis against double-distilled water, to yieldbarnase-ubiquitin fusion protein that is approximately 95% pure asjudged by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0081] To confirm the switching mechanism of the model for the MEDFmolecular switch as embodied in the exemplary ubiquitin-barnase fusionprotein, and to characterized its structure, stability, and enzymaticfunction, experiments were performed by the inventors herein upon thepurified ubiquitin-barnase fusion protein, obtained as describedhereinabove All experiments were performed in 10 mM potassium phosphate(pH 7.5), 0.1 M NaCl. The circular dichroism (“CD”) spectra are shown inFIG. 3 as a function of temperature. In FIG. 3., circles and squaresindicate 5° C. and 50° C., respectively; other scans were recorded at 5°C. increments between these two limits. Below 20° C., thebarnase-ubiquitin fusion protein exhibits molar ellipticities andspectral features nearly identical to those of barnase. A particularlydiagnostic characteristic of barnase that is also observed for thebarnase-ubiquitin fusion protein is a minimum at 231 nm attributed toTrp94. As temperature is increased, however, the spectrum shifts to onethat strongly resembles ubiquitin. The 231 nm minimum disappears, andthe position and molar ellipticity of the new minimum are consistentwith native ubiquitin FIG. 4 shows a graph of the conversion aubiquitin-barnase fusion protein from the barnase conformation to theubiquitin conformation, monitored by ellipticity at 230 nm (opentriangles, solid line) and barnase enzymatic activity (closed triangles,dashed line). Lines are for illustrative purpose only. Activities weredetermined by recording initial velocities in triplicate (2 mMubiquitin-barnase fusion protein, 50 mM guanylyl(3-5) uridine3-monophosphate (“GpUp”) ) and are normalized to the largest value. Ascan be seen from FIG. 4, the transition is fully reversible and has amidpoint of about 30 degrees C. Further, it appears to be two-state; anisodichroic point near 223 nm is apparent, and plotting the transitionat different wavelengths yields identical midpoints.

[0082] The temperature-induced transition from barnase to ubiquitin isconsistent with the higher thermal stability of ubiquitin. Ubiquitin andbarnase unfold with midpoints of about 100 degrees C. (pH 7) and 55degrees C. (pH 6.3) respectively. The possibility that the ubiquitin andbarnase domains are simultaneously folded is ruled out by theobservation that the molar ellipticity of the ubiquitin-barnase fusionprotein never exceeds that of the individual proteins at any wavelengthor temperature.

[0083] To further test the mutual exclusivity of ubiquitin and barnasedomain folding, the inventors herein monitored urea-induced denaturationby Circular dichroism and Trp fluorescence. ubiquitin and barnasecontain zero and three Trp residues, respectively. Fluorescence istherefore expected to report primarily on structural changes within thebarnase domain, whereas Circular dichroism reports on both domains. FIG.5 is a graph showing the denaturation of the barnase domain (10 degreesC., circles) and the ubiquitin domain (40 degrees C., squares) inducedby urea and guanidine hydrochloride (“GdnHCl”), respectively. Data werecollected by Circular dichroism at 230 nm (open symbols, black lines) orby Trp fluorescence at 320 nm (closed symbols, grey line). Linesrepresent best fits to the linear extrapolation equation. At 10 degreesC., the Circular dichroism and fluorescence curves reveal a singlecooperative unfolding transition. It appears to be two-state;thermodynamic parameters obtained by fitting both data sets to thelinear extrapolation equation are identical within error (DG=4.1±0.2kcal×mol₋₁, m=2.2±0.1 kcal×mol₋₁×M⁻¹, Cm=2.3±0.05 M). The fact that onlyone transition is apparent by Circular dichroism confirms that only onedomain is folded. The agreement between Circular dichroism andfluorescence curves indicates that this domain is barnase.

[0084] If the situation is reversed by raising temperature above 30degrees C., addition of denaturant at 40 degrees C. is predicted togenerate an unfolding transition by Circular dichroism but no transitionby fluorescence. 6 M urea failed to produce a change in either spectrum.Addition of the stronger denaturant guanidine hydrochloride, however,yields the expected Circular dichroism unfolding transition with thefollowing thermodynamic parameters: DG=8.5±0.1 kcal×mol⁻¹, m=2.5±0.1kcal×mol⁻¹×M⁻¹, C_(m)=3.4±0.05 M, as shown in FIG. 5.

[0085] In contrast to molar ellipticity, fluorescence emission at 320 nmdoes not change significantly as a function of guanidine hydrochlorideconcentration, suggesting that the barnase Trp residues are solventexposed at all denaturant concentrations. This conclusion is supportedby the finding that the wavelength of maximum emission remains constantat 356 nm, the value for unfolded barnase. At 10 degrees C., thiswavelength shifts from 340 nm in the absence of denaturant to 356 nm in6 M urea. These data prove that:

[0086] [i] folding of the barnase domain induces unfolding of theUbiquitin domain, and vice versa; and,

[0087] [ii] temperature provides an efficient switch between the twofolded conformations of the ubiquitin-barnase fuision protein

[0088] To determine how tightly folding of the ubiquitin domain iscoupled to unfolding of the barnase domain, the inventors hereinmeasured the enzymatic activity of the ubiquitin-barnase fusion proteinas a function of temperature, using the substrate guanylyl(3′-5′)uridine3′-monophosphate. As shown in FIG. 4, loss of The barnase-ubiquitinfusion protein activity in the ubiquitin-barnase fusion protein mirrorsthe structural conversion from barnase to ubiquitin, although theapparent midpoint of the former transition occurs at a highertemperature.

[0089] A likely reason is that substrate binding in the enzyme assayspreferentially stabilizes the barnase domain. The complete loss ofbarnase activity above 50 degrees C., together with the molarellipticity values shown in FIG. 3, provides strong evidence thatubiquitin domain folding induces complete unfolding of the barnasedomain.

[0090] The preceding experimental results suggested to the inventorsherein that temperature can be used to regulate cytotoxicity of theubiquitin-barnase fusion protein in vivo. The inventors herein testedthis prediction by transforming E. coli with a plasmid containing theubiquitin-barnase fusion protein gene under control of an isopropylb-D-thiogalactopyranoside (“IPTG”) -induced T7 promoter. Like barnase,the ubiquitin-barnase fuision protein is extremely lethal. The plasmidwas found to be unstable in all strains of E. coli, including those thatdo not harbor the T7 RNA polymerase gene. Very few transformants wereconsistently recovered, and in each case the ubiquitin-barnase fusionprotein gene was found to contain frameshift or nonsense mutations inthe barnase coding region.

[0091] To overcome this problem, the inventors herein inserted thebarstar gene and its natural promoter from Bacillus amyloliquifaciensinto the foregoing plasmid, thereby creating a pETMT plasmid that isstable in E. coli.

[0092] As shown in Table 1 hereinbelow, wherein figures are the averageswith standard deviations are obtained from five plates, far fewercolonies were obtained when plates were grown at 15 degrees C. comparedto 37 degrees C. Moreover, 36 mg/m L IPTG was sufficient to kill nearlyall of the bacteria at 15 degrees C., whereas 143 mg/mL IPTG wasrequired to achieve a comparable result at 37 degrees C.

We claim:
 1. A model for a mutually exclusive folding domain molecularswitch including a fusion protein comprising at least one insert proteinhaving an insert domain lying between an amino terminal and a carboxylterminal of said at least one insert protein, said insert domain beingassociated with a first quantity of free energy; and, a target proteinhaving at least one surface loop that begins at an alpha carbon of afirst surface loop amino acid and terminates at an alpha carbon of asecond surface loop amino acid, said at least one surface loopcomprising a target domain of said target protein, said target domainbeing associated with a second quantity of free energy, wherein, said atleast one insert protein is operatively inserted within said at leastone surface loop between said alpha carbon of said first surface loopamino acid and said alpha carbon of said second surface loop amino acidsuch that an amino-carboxyl length extending between an alpha carbon ofsaid amino terminal of said at least one insert protein and an alphacarbon of said carboxyl terminal of said at least one insert protein isat least two-times greater than an alpha-carbon-alpha-carbon lengthextending between said alpha carbon of said first surface loop aminoacid and said alpha carbon of said second surface loop amino acid. 2.The fusion protein of claim 1, wherein said insert domain exists ineither a folded or unfolded conformation and said target domain existsin either a folded or unfolded conformation, said insert domain and saidtarget domain comprising a cooperative and reversible conformationalequilibrium such that if said insert domain is in its foldedconformation, said at least one target domain is in its unfoldedconformation and vice versa.
 3. The fusion protein of claim 2, whereinall or part of said first quantity of free energy is made available todrive a folding of said target domain from its unfolded conformation bymeans of a first controllable effector signal, and all or part of saidsecond quantity of free energy is made available to drive a folding ofsaid insert domain from its unfolded conformation by means of a secondcontrollable effector signal
 4. The fusion protein of claim 3, whereinsaid first controllable effector signal is selected from the groupcomprising ligand binding, pH, temperature, chemical denaturants, ormutations in either said insert domain or said target domain.
 5. Thefusion protein of claim 3, wherein said second controllable effectorsignal is selected from the group comprising ligand binding, pH,temperature, chemical denaturants, or mutations in either said insertdomain or said target domain.
 6. The fusion protein of claim 2, whereinsaid insert domain and said target domain are disenabled fromsimultaneously co-existing in their respective folded conformations. 7.The fusion protein of claim 2, wherein said insert domain and saidtarget domain are disenabled from simultaneously co-existing in theirrespective unfolded conformations.
 8. The fusion protein of claim 2,wherein any excess of said first quantity of free energy of said insertdomain that is not necessary to stabilize said insert domain in itsfolded conformation is spontaneously transferred, through the structureof said fusion protein, to said target domain to unfold it from itsfolded conformation.
 9. The fusion protein of claim 2, wherein anyexcess of said second quantity of free energy of said target domain thatis not necessary to stabilize said target domain in its foldedconformation is spontaneously transferred, through the structure of saidfusion protein, to said insert domain to unfold it from its foldedconformation.
 10. The fusion protein of claim 2, wherein said at leastone insert protein comprises human ubiquitin, said insert domaincomprises a regulatory domain of human ubiquitin, said target proteincomprises barnase, said at least one target domain comprises a cytotoxicdomain of barnase, said amino-carboxyl length is about 38 Å, said firstsurface loop amino acid comprises proline in the number 64 position(“Pro64”), said second surface loop amino acid comprises threonine inthe number 70 position (“Thr70”), and said alpha-carbon-alpha-carbonlength is about 10.4 Å.
 11. The fusion protein of claim 10 wherein saidregulatory domain of human ubiquitin and said cytotoxic domain ofbarnase comprise a cooperative and reversible conformationalequilibrium, that may be determined by said controllable first andsecond effector signals.
 12. A method for the production of a proteincomprising the steps of: a. selecting a linker containing first andsecond restriction sites between a Lys66 and a Ser67 codon of a barnasegene; b. using said first and second restriction sites of said linker tooperationally insert a ubiquitin gene between two amino-acid codons ofsaid linker, thereby creating a ubiquitin-barnase fusion gene; c. fullysequencing said ubiquitin-barnase fusion gene to verify its integrity;d. using enzymes to operationally insert said ubiquitin-barnase fusiongene into any plasmid of a BL21 (DE3) family, thereby creating aninterim ubiquitin-barnase fusion expression plasmid; e. operationallyinserting a gene for barstar and its natural promoter from Bacillusamyloliquifaciens into said interim ubiquitin-barnase fusion expressionplasmid, thereby creating a ubiquitin-barnase fusion-barstar complexplasmid; f. cloning said gene for barstar into a T7 promoter-containingplasmid conferring resistance to an antibiotic other than ampicillinonto cells transformed by said T7 promoter-containing plasmid, therebycreating a barstar plasmid; g. transforming E. coli BL21 (DE3) cellsgrown at about 20 to 37 degrees C. in any medium compatible with E. coligrowth using both said barstar plasmid and said ubiquitin-barnasefusion-barstar complex plasmid, and inducing said E. coli BL21 (DE3)cells with about 100 mg/L isopropyl b-D-thiogalactopyranoside; h.harvesting said transformed E. coli cells by centrifugation after about2 to 12 hours; after said induction; i. placing said harvested E. colicells in 10 mM sodium phosphate at a pH of 7.5, thereby creating asolution of harvested E. coli cells; j. lysing said solution ofharvested E. coli cells by repeated freeze-thaw cycles in the presenceof about 10 mg/liter lysozyme, thereby creating a lysate; k. addingabout 10 mg/liter DNase I to reduce the viscosity of said lysate; l.centrifuging said reduced viscosity lysate to remove insolubles, therebyforming a supernatant; m. adding about 8 M urea to said supernatant todissociate bound barstar; n. Removing said dissociated barstar from saidsupernatant by passing said supernatant through an anion exchangechromatography resin to yield a solution; o. loading said solution ontoa cation exchange column; p. washing said solution with about 10 mMsodium phosphate (pH about 7.5) and about 6 M urea; q. eluting saidsolution using a 0 to 0.2 M NaCl gradient; r. Removing said urea fromsaid dilution by dialysis against double-distilled water to yieldbarnase-ubiquitin fusion protein.